U.S. patent number 10,689,444 [Application Number 15/416,513] was granted by the patent office on 2020-06-23 for recombinant monovalent antibodies.
This patent grant is currently assigned to Institut National de la Sante et de la Recherche Medicale, OSE Immunotherapeutics. The grantee listed for this patent is Institut National de la Sante et de la Recherche Medicale (INSERM), OSE Immunotherapeutics. Invention is credited to Flora Coulon, Caroline Mary, Bernard Vanhove.
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United States Patent |
10,689,444 |
Vanhove , et al. |
June 23, 2020 |
Recombinant monovalent antibodies
Abstract
The invention relates to recombinant monovalent antibodies which
are heterodimers of a first protein chain comprising the variable
domain of the heavy chain of an antibody of interest and the CH2
and CH3 domains of an IgG immunoglobulin and a second protein chain
comprising the variable domain of the light chain of said
immunoglobulin of interest and the CH2 and CH3 domains of said IgG
immunoglobulin. These antibodies can be used in particular as
therapeutic agents in all cases where monovalent binding to a
ligand such a cellular receptor is required.
Inventors: |
Vanhove; Bernard (Reze,
FR), Mary; Caroline (Sainte Pazanne, FR),
Coulon; Flora (Saint Georges de Montaigu, FR) |
Applicant: |
Name |
City |
State |
Country |
Type |
OSE Immunotherapeutics
Institut National de la Sante et de la Recherche Medicale
(INSERM) |
Nantes
Paris |
N/A
N/A |
FR
FR |
|
|
Assignee: |
OSE Immunotherapeutics (Nantes,
FR)
Institut National de la Sante et de la Recherche Medicale
(Paris, FR)
|
Family
ID: |
40435756 |
Appl.
No.: |
15/416,513 |
Filed: |
January 26, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170166643 A1 |
Jun 15, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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13144471 |
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9587023 |
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PCT/IB2010/000196 |
Jan 13, 2010 |
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Foreign Application Priority Data
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Jan 14, 2009 [EP] |
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09290029 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
16/2818 (20130101); A61P 37/00 (20180101); A61P
37/08 (20180101); A61P 29/00 (20180101); A61P
37/06 (20180101); C07K 2317/565 (20130101); C07K
2317/74 (20130101); C07K 2317/524 (20130101); C07K
2319/00 (20130101); Y02A 50/41 (20180101); C07K
2317/94 (20130101); C07K 2317/52 (20130101); A61K
2039/505 (20130101); C07K 2317/35 (20130101); C07K
2318/10 (20130101); C07K 2317/526 (20130101); C07K
2317/14 (20130101); Y02A 50/30 (20180101); C07K
2317/56 (20130101) |
Current International
Class: |
C07K
16/28 (20060101); A61K 39/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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02/051871 |
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Jul 2002 |
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WO |
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2004/058820 |
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Jul 2004 |
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WO |
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2005/063816 |
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Jul 2005 |
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WO |
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2007/048037 |
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Apr 2007 |
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WO |
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2007/087673 |
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Aug 2007 |
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WO |
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2008/145138 |
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Dec 2008 |
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WO |
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Other References
Webber et al., "Preparation and Characterization of a
Disulfide-Stabilized Fv Fragment of the Anti-Tac Antibody:
Comparison with its Single-Chain Analog," Molecular Immunology, 32:
249-258 (1995). cited by applicant .
Jain et al., "Engineering antibodies for clinical applications,"
Trends in Biotechnology, 25: 307-316 (2007). cited by applicant
.
Paul ed., "Fundamental Immunology: Immunogenicity and Antigen
Structure," 242 (1993). cited by applicant .
Labrijn et al., "When binding is enough: nonactivating antibody
formats," Current Opinion in Immunology, 20: 479-485 (2008). cited
by applicant.
|
Primary Examiner: Duffy; Brad
Attorney, Agent or Firm: Morgan, Lewis & Bockius LLP
Claims
The invention claimed is:
1. A recombinant antibody derived from a parent antibody directed
against an antigen of interest, wherein said recombinant antibody
is an heterodimer of: i. a first protein chain consisting
essentially of, from its N-terminus to its C-terminus: a. a region
A having the structure of the variable domain of the heavy chain of
an immunoglobulin, said region A comprising the CDRs of the heavy
chain of said parent antibody; b. a region B consisting of a
peptide linker and the CH2 and CH3 domains of an IgG
immunoglobulin, wherein said peptide linker comprises one or more
cysteine residues; ii. a second protein chain consisting
essentially of, from its N-terminus to its C-terminus: a. a region
A' having the structure of the variable domain of the light chain
of an immunoglobulin, said region A' comprising the CDRs of the
light chain of said parent antibody; b. a region B identical to the
region B of the first polypeptide; wherein said first and second
protein chains are devoid of a hinge region or any portion thereof
and of a CH1 domain of an IgG immunoglobulin, and the first and
second protein chains are linked by at least one inter-chain
disulfide bond.
2. A recombinant monovalent antibody of claim 1, wherein the
peptide linker is a peptide sequence of 1 to 16 amino acids.
3. A recombinant antibody of claim 1, wherein the CH2 and CH3
domains are those of an immunoglobulin of the IgG1 subclass, or of
the IgG4 subclass.
4. A recombinant monovalent antibody of claim 1, wherein the region
A consists of the variable domain of the heavy chain of the parent
antibody.
5. A recombinant monovalent antibody of claim 1, wherein the region
A' consists of the variable domain of the light chain of the parent
antibody.
6. A recombinant monovalent antibody of claim 1, wherein the parent
antibody is the monoclonal immunoglobulin CD28.3, produced by the
hybridoma deposited at Collection Nationale de Cultures de
Microorganismes under Accession No. CNCM I-2582.
7. A polynucleotide selected from the group consisting of: (a) a
polynucleotide comprising a sequence encoding the first protein
chain of a recombinant monovalent antibody according to claim 1;
and (b) a polynucleotide comprising a sequence encoding the second
protein chain of a recombinant monovalent antibody according to
claim 1.
8. An expression vector comprising a polynucleotide of claim 7.
9. A cell transformed with a polynucleotide (a) and a
polynucleotide (b) of claim 7.
10. A method for preparing a recombinant monovalent antibody,
wherein said method comprises culturing the transformed cell of
claim 9, and recovering said recombinant monovalent antibody from
said culture.
11. A medicinal product comprising the recombinant antibody of
claim 1.
Description
SEQUENCE LISTING SUBMISSION VIA EFS-WEB
A computer readable text file, entitled "SequenceListing.txt,"
created on or about Jul. 13, 2011 with a file size of about 23 kb
contains the sequence listing for this application and is hereby
incorporated by reference in its entirety.
The invention relates to recombinant monovalent antibodies, in
particular IgG antibodies, and to their therapeutic uses.
An antibody (immunoglobulin) molecule is a Y-shaped tetrameric
protein composed of two heavy (H) and two light (L) polypeptide
chains held together by covalent disulfide bonds and noncovalent
interactions.
Each light chain is composed of one variable domain (VL) and one
constant domain (CL). Each heavy chain has one variable domain (VH)
and a constant region, which in the case of IgG, IgA, and IgD,
comprises three domains termed CH1, CH2, and CH3 (IgM and IgE have
a fourth domain, CH4). In IgG, IgA, and IgD classes the CH1 and CH2
domains are separated by a flexible hinge region, which is a
proline and cysteine rich segment of variable length (generally
from about 10 to about 60 amino acids in IgG).
The variable domains show considerable variation in amino acid
composition from one antibody to another. Each of the VH and the VL
variable domains comprises three regions of extreme variability,
which are termed the complementarity-determining regions (CDRs),
separated by less variable regions called the framework regions
(FRs). The non-covalent association between the VH and the VL
region forms the Fv fragment (for "fragment variable") which
contains one of the two antigen-binding sites of the antibody. ScFv
fragments (for single chain fragment variable), which can be
obtained by genetic engineering, associates in a single polypeptide
chain the VH and the VL region of an antibody, separated by a
peptide linker.
Other functional immunoglobulin fragments can be obtained by
proteolytic fragmentation of the immunoglobulin molecule. Papain
treatment splits the molecule into three fragments: two
heterodimeric Fab fragments (for `fragment antigen binding`), each
associating the VL and CL domains of the light chain with the VH
and CH1 domains of the heavy chain, and one homodimeric Fc fragment
(for "fragment crystalline"), which comprises the CH2 and CH3 (and
eventually CH4) domains of the light chain. Pepsin treatment
produces the F(ab)'2 fragment which associates two Fab fragments,
and several small fragments.
The Fc fragment does not bind the antigen, but is responsible for
the effector functions of the antibody, including in particular
binding to Fc receptors and complement fixation. The Fv, Fab, and
F(ab)'2 fragments retain the antigen-binding ability of the whole
antibody. However, the F(ab)'2 fragments, like the whole
immunoglobumin molecule, are divalent (i.e. they contain two
antigen binding sites and can bind and precipitate the antigen),
while the Fv and Fab fragments are monovalent (they contain one
antigen binding site, and can bind but cannot precipitate the
antigen).
Antibodies directed against cell-surface receptors are of great
interest for the development of therapeutic agents for various
disorders and diseases. They are generally used for their
properties to mimic the structure of a biological ligand of a
target receptor. In some cases this structural similarity may
result in agonistic effects leading to the activation of the target
receptor; in other cases it may result in antagonistic effects,
leading to the blocking of the target receptor.
However, many antibodies having antagonistic properties when used
as monovalent fragments may also show agonistic effects when used
as full length antibodies. These agonistic effects result from the
bivalency of the full-length antibodies, which induces the
crosslinking of the target receptors on the cell surface, leading
to receptor activation. This phenomenon is unwanted when the
desired therapeutic activity relies upon an antagonistic effect.
Examples of receptors that are activated by crosslinking include
CD28, CD3 (DAMLE et al., J. Immunol., 140, 1753-61, 1988; ROUTLEDGE
et al., Eur J Immunol, 21, 2717-25, 1991), TNF receptors, etc . . .
.
The monovalent forms of antagonistic antibodies, such as Fab or
scFv fragments, are devoid of agonistic activity. Therefore, they
are useful therapeutic agents to block a cell receptor without
inducing its cross-linking. However, their therapeutic use is
hampered by their short half-life in vivo; they are eliminated
within minutes and would require a continuous administration. To
overcome this problem, it has been proposed to fuse these
monovalent fragments with large molecules such as water-soluble
proteins (PCT WO02051871) or polyethylene glycol (BLICK &
CURRAN, BioDrugs, 21, 195-201; discussion 02-3, 2007).
Another approach for producing monovalent antibodies has been to
construct fusion proteins associating one Fab fragment (i.e an
heterodimer comprising the VL and the CL regions of the light
chain, and the VH and the CH1 region of the heavy chain) with one
Fc fragment (i.e an homodimer comprising the CH2 and CH3 regions of
the heavy chains). ROUTLEDGE et al. (ROUTLEDGE et al., Eur J
Immunol, 21, 2717-25, 1991) describe the construction of a
monovalent antibody by introduction into an antibody-producing cell
of a truncated Ig heavy chain gene encoding only the hinge, CH2 and
CH3 domains; the expression of this gene in the antibody producing
cell results in N-terminally truncated heavy chains (devoid of the
VH and CH1 domains) which can either associate between them to form
Fc molecules, or with full length heavy chains produced by the
antibody producing cell to form a monovalent antibody molecules
comprising a full-length light chain, a full-length heavy chain,
and a N-terminally truncated heavy chain. PCT WO 2007/048037
describes monovalent antibodies which are heterodimers resulting
from the association of an immunoglobulin heavy chain with a fusion
protein comprising an immunoglobulin light chain and a Fc
molecule.
An advantage of this approach is that the resulting antibodies
contain an IgG Fc domain, which in some cases, is useful if one
desires to retain some of the effector functions of the IgG
molecule, and which also allows to target the molecule to the
neonatal Fc receptor (FcRn) expressed by endothelial cells. This
receptor actively traps several macromolecules, including
antibodies, inside the blood stream conferring them an extended
serum half-live. The binding of IgG molecules to this receptor
facilitates their transport, and allows their protection from
degradation.
The IgG Fc domain of immunoglobulins has also been utilized to form
fusion proteins with molecules other than antibodies, for instance
cytokines, growth factors, soluble growth factors, allowing to
extend their half-life in the bloodstream, and also to deliver them
by non-invasive routes, for instance by pulmonary administration
(DUMONT et al., BioDrugs, 20, 151-60, 2006).
In the case of monovalent antibodies, the fusion proteins
containing the IgG Fc domain which have been described until now
also comprise the CL and/or the CH1 region. It is generally
believed that these regions, which are part of the Fab fragment,
play an important part in the correct assembly of the IgG molecule,
and can also influence the antigen/antibody interaction.
As indicated above, one of the cell surface receptors known to be
stimulated after its engagement by bivalent antibodies, and which
can be efficiently blocked by certain monovalent fragments of some
antibodies, is the CD28 receptor. By way of example, it has been
shown that it was possible to efficiently block CD28 with Fab
fragments or with a fusion protein comprising a scFv fragment of
the anti-CD28 monoclonal antibody CD28.3, fused with
alphal-antitrypsin (VANHOVE et al., Blood, 102, 564-70, 2003). This
approach demonstrated an efficacy in vitro as well as in organ
transplantation in mice and in primates (POIRIER et al., World
Transplant Congress, Sydney, Australia. Aug. 16-21, 2008).
The inventors have sought to further improve the pharmacokinetics
properties of monovalent fragments of CD28.3. With this purpose,
they have first attempted to construct a recombinant monovalent
antibody similar to those disclosed in the prior art, by fusing the
each of the VH and VL domains of CD28.3 to the CH1-CH2-CH3 domains
of an heterologous IgG molecule. However this attempt failed to
result in a protein with the required antibody activity.
The inventors then tried to remove the CH1 domains of these fusion
proteins and found that the resulting monovalent antibody was
secreted and active, and that it behaves in vitro like its
corresponding Fab fragment. Further, after intravenous injection in
mice, it showed an elimination half-live that was significantly
longer than Fab fragments and not significantly different from IgG
antibodies.
These results show that combining the variable domains of a
monoclonal antibody with only the CH2-CH3 domains rather than with
all the constant domains of an IgG molecule allows to obtain a
functional monovalent antibody, having the prolonged in vivo
half-live that is conferred by the presence of an Fc fragment. This
format can be used to generate therapeutic antibodies in all cases
where monovalent binding to a ligand, for instance a cellular
receptor, is required.
Therefore, an object of the present invention is a recombinant
monovalent antibody derived from a parent antibody directed against
an antigen of interest, wherein said recombinant antibody is an
heterodimer of:
a first protein chain consisting essentially of, from its
N-terminus to its C-terminus:
a region A having the structure of the VH domain of an
immunoglobulin, said region A comprising the CDRs of the heavy
chain of said parent antibody;
a region B consisting of a peptide linker and the CH2 and CH3
domains of an IgG immunoglobulin;
a second protein chain consisting essentially of, from its
N-terminus to its C-terminus:
a region A' having the structure of the VL domain of an
immunoglobulin, said region A' comprising the CDRs of the light
chain of said parent antibody;
a region B identical to the region B of the first polypeptide.
The parent antibody can be any antibody directed against the
antigen of interest; it can be a native monoclonal antibody; it can
also be a recombinant or synthetic antibody, such as a chimeric
antibody, a humanized antibody, or an antibody originating from
phage-display or ribosome display technologies.
A region having the structure of the VH or of the VL domain of an
immunoglobulin comprises, as indicated above, four framework
regions (FRs), connected by three hypervariable regions or
complementarity determining regions (CDRs) which are involved in
antigen recognition specificity. In a recombinant monovalent
antibody of the invention, regions A and A' can consist of the
native VH or VL domains of the parent antibody; however, they can
also be obtained by incorporating the CDRs of the parent antibody
into the framework regions (FRs) of another antibody, in particular
of an antibody of human origin, using techniques, known in
themselves, of CDR grafting.
The peptide linker of region B may comprises from 0 to 16 amino
acids. It comprises preferably 5 to 7 amino acids. Examples of
suitable peptide linkers are those which are used in the
construction of scFv fragments, such are those disclosed for
instance by FREUND et al. (FEBS Lett. 320, 97-100, 1993) or by SHAN
et al. (J Immunol. 162, 6589-95, 1999).
Said peptide linker may be devoid of cysteine residues, or may
comprise one or more cysteine residue(s). A peptide linker devoid
of cysteine residues will be preferred if the monovalent antibody
is to be produced in the E. coli periplasm. An example of a peptide
linker devoid of cysteine residues is a peptide having the sequence
TVAAPS (SEQ ID NO: 5).
Alternatively, a peptide linker comprising cysteine residues allows
the formation of inter-chain disulfide bonds, which help to
stabilize the heterodimer. As a peptide linker comprising cysteine
residues, one can use for instance the hinge region of a naturally
occurring IgG. A preferred hinge region is the hinge region of IgG2
immunoglobulins having the sequence ERKCCVECPPCP (SEQ ID NO: 12),
which provides a high stability.
The CH2 and CH3 domains are preferably those of an immunoglobulin
of human origin of the IgG isotype. Said IgG can belong to any of
the IgG subclasses (IgG1, IgG2, IgG3 or IgG4). Preferably, it
belongs to the IgG1 subclass or the IgG4 subclass.
Besides the essential constituents listed above, the first and/or
the second protein chain can further comprise one or more optional
polypeptide sequence(s) which is (are) not involved in the
biological properties of the recombinant monovalent antibody, but
may facilitate its detection or purification. For instance said
polypeptide sequence can be a tag polypeptide, such as a
streptavidin-binding peptide, an hexa-histidine (His.sub.6) tag, or
a FLAG-tag.
The first and/or the second protein chain can be glycosylated or
not.
According to a particular embodiment of the invention, the parent
antibody is the monoclonal antibody CD28.3, produced by the
hybridoma CNCM 1-2582. The hybridoma CNCM 1-2582 is disclosed in
PCT WO02051871, and has been deposited, according to the terms of
the Treaty of Budapest, on Nov. 28, 2000, with the CNCM (Collection
Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux,
75724 PARIS CEDEX 15).
A particular example of a recombinant monovalent antibody of the
invention, which is described in detail in the Examples below, is
an antibody wherein the polypeptide sequence of the first protein
chain is SEQ ID NO: 2, and the polypeptide sequence of the second
protein chain is SEQ ID NO: 4. Another example of a recombinant
monovalent antibody of the invention, is an antibody wherein the
polypeptide sequence of the first protein chain is SEQ ID NO: 13,
and the polypeptide sequence of the second protein chain is SEQ ID
NO: 14.
Another object of the invention is a polynucleotide comprising a
sequence encoding the first protein chain and/or a sequence
encoding the second protein chain of a recombinant monovalent
antibody of the invention. Said polynucleotides may also comprise
additional sequences: for instance they may advantageously comprise
a sequence encoding a leader sequence or signal peptide allowing
secretion of said protein chain. They may optionally also comprise
one or more sequence(s) encoding one or more tag
polypeptide(s).
The present invention also encompasses recombinant vectors, in
particular expression vectors, comprising a polynucleotide of the
invention, associated with transcription- and
translation-controlling elements which are active in the host cell
chosen. Vectors which can be used to construct expression vectors
in accordance with the invention are known in themselves, and will
be chosen in particular as a function of the host cell intended to
be used.
The present invention also encompasses host-cells transformed with
a polynucleotide of the invention. Preferably, said host cell is
transformed with a polynucleotide comprising a sequence encoding
the first protein chain of a recombinant monovalent antibody of the
invention and a polynucleotide comprising a sequence encoding the
second protein chain of a recombinant monovalent antibody of the
invention, and expresses said recombinant antibody. Said
polynucleotides can be inserted in the same expression vector, or
in two separate expression vectors.
Host cells which can be used in the context of the present
invention can be prokaryotic or eukaryotic cells. Among the
eukaryotic cells which can be used, mention will in particular be
made of plant cells, cells from yeast, such as Saccharomyces,
insect cells, such as Drosophila or Spodoptera cells, and mammalian
cells such as HeLa, CHO, 3T3, C127, BHK, COS, etc., cells.
The construction of expression vectors of the invention and the
transformation of the host cells can be carried out by the
conventional techniques of molecular biology.
Still another objet of the invention is a method for preparing a
recombinant monovalent antibody of the invention, Said method
comprises culturing an host-cell transformed with a polynucleotide
comprising a sequence encoding the first protein chain of a
recombinant monovalent antibody of the invention, and with a
polynucleotide comprising a sequence encoding the second protein
chain of a recombinant monovalent antibody of the invention, and
recovering said recombinant monovalent antibody from said
culture.
If the protein is secreted by the host-cell, it can be recovered
directly from the culture medium; if not, cell lysis will be
carried out beforehand. The protein can then be purified from the
culture medium or from the cell lysate, by conventional procedures,
known in themselves to those skilled in the art, for example by
fractionated precipitation, in particular precipitation with
ammonium sulfate, electrophoresis, gel filtration, affinity
chromatography, etc.
A subject of the invention is also a method for producing a protein
in accordance with the invention, characterized in that it
comprises culturing at least one cell in accordance with the
invention, and recovering said protein from said culture.
The recombinant monovalent antibodies of the invention can be used
to obtain medicinal products. These medicinal products are also
part of the object of the invention.
For instance, recombinant monovalent antibodies of the invention
derived from the parent antibody CD28.3 can be used to obtain
immunosuppressant medicinal products which selectively blocks T
cell activation phenomena involving the CD28 receptor. Such
immunosuppressant medicinal products which act by selective
blocking of CD28 have applications in all T lymphocyte-dependent
pathological conditions, including in particular transplant
rejection, graft-versus-host disease, T lymphocyte-mediated
autoimmune diseases, such as type I diabetes, rheumatoid arthritis
or multiple sclerosis, and type IV hypersensitivity, which is
involved in allergic phenomena and also in the pathogenesis of
chronic inflammatory diseases, in particular following infection
with a pathogenic agent (in particular leprosy, tuberculosis,
leishmaniasis, listeriosis, etc.).
The present invention will be understood more clearly from the
further description which follows, which refers to nonlimiting
examples of the preparation and properties of a recombinant
monovalent antibody (hereafter referred to as Mono28Fc) in
accordance with the invention.
LEGENDS OF THE DRAWINGS
FIG. 1A: Nucleotidic and amino acid sequence of Mono28Fc, VH-CH2CH3
chain.
Underlined: VH domain. Bold: linker. Double underlining: IgG1
CH2-CH3 domains.
FIG. 1B: Nucleotidic and amino acid sequence of Mono28Fc, VL-CH2CH3
chain.
Underlined: VL domain. Bold: linker. Double underlining: IgG1
CH2-CH3 domains.
FIG. 1C: Molecular constructions allowing the expression of
Mono28Fc after transfection into eukaryotic host cells.
pCMV: promoter of the cytomegalovirus. Igk leader: signal sequence
from the mouse immunoglobulin kappa light chain. VH: variable
domain of the heavy chain of the CD28.3 antibody. VL: variable
domain of the light chain of the CD28.3 antibody. CH2 and CH3
represent the corresponding domains of the IgG1 human
immunoglobulin.
FIG. 1D: Expression plasmids for the synthesis of Mono28Fc in
eukaryotic cells.
A: plasmid for the synthesis of the VH(Hc)-CH2-CH3 protein. B:
plasmid for the synthesis of the VL(Lc)-CH2-CH3 protein. pCMV:
promoter of the cytomegalovirus. Igk leader: signal sequence from
the mouse immunoglobulin kappa light chain. Hc: VL variable domain
of the heavy chain of the CD28.3 antibody. Lc: VL variable domain
of the light chain of the CD28.3 antibody. CH2 and CH3 represent
the corresponding domains of the IgG1 human immunoglobulin. BGH pA:
signal for the initiation of the 3' polyadenylation of the mRNA
molecule, from the bovine growth hormone. Zeocin, ampicillin:
resistance genes for the corresponding antibiotic.
FIG. 2: Western blot analysis of pSecVHFc and pSecVLFc
expression.
A: Supernatants from Cos cells transfected with the indicated
plasmids were collected and reduced before analysis by 10 min.
incubation at 100.degree. C. with 10 mM DTT. B: no reduction.
Molecular weights are indicated on the left sides.
FIG. 3: Activity ELISA. Recombinant CD28 was immobilized on
microtitration plates.
A: Supernatants from control, transfected or co-transfected Cos
cells were added at the indicated dilutions, washed and revealed
with rabbit anti-VHNL antibodies plus anti-rabbit
immunoglobulins-HRP. GFP: negative control; transfection with an
irrelevant GFP plasmid. Sc28AT: positive control; transfection with
a plasmid coding for a single-chain Fv against CD28. VLFc:
transfection with the pSec-VLFc plasmid. VHFc: transfection with
the pSec-VHFc plasmid. VH-(CH2-CH3)+VL-CH2-CH3: co-transfection
with the pSec-VLFc and the pSec-VHFc plasmids. B: Binding ELISA on
recombinant CD28 of purified Mono28Fc molecules at the indicated
concentration. Revelation is as in A. Dots are means of
triplicates.
FIG. 4: Flow cytometry.
CD28.sup.+ Jurkat T cells and CD28.sup.- Raji B cells were
incubated with purified Mono28Fc or with CD28.3 Fab fragments at 10
.mu.g/ml for 30 min. at 4.degree. C., washed and revealed with
rabbit anti-VHNH antibodies plus FITC-labeled goat anti-rabbit
immunoglobulins (black profiles). As a control, cells were
incubated with rabbit anti-VHNH antibodies plus FITC-labeled goat
anti-rabbit immunoglobulins only (grey profiles). Cells were then
washed, fixed and analyzed by Facs.
FIG. 5: Activation assay.
Human PBMC (10.sup.5/well) were cultivated in medium or in medium
plus 10 .mu.g/ml Mono28Fc, sc28AT monovalent antibodies or with
ANC28.1 superagonist antibodies for 3 days. 0.5 .mu.Ci
.sup.3H-tymidine was added for the last 16 h of the culture.
Incorporated radioactivity was evaluated on a scintillation counter
after transfer on nitrocellulose membranes.
FIG. 6: Pharmacokinetic in mice.
A: Indicated proteins were injected i.v. into swiss mice and blood
samples were collected after the indicated time points. CD28
binding activity was measured by ELISA. N=4 for each point, dots
are means of the 4 measurements. B: Elimination half-lives
(T.sub.1/2.beta.) were calculated from the curves in A.
FIG. 7: Molecular constructions combining VH, VL with CH1-CH2-CH3
are non-functional.
A: RT-PCR analysis of the VH and VL mRNA chains expression after
transfection of Cos cells. B: Western blot analysis of supernatants
(right panel) and lysates (left panel) of Cos cell transfected with
pSecVH-CH1-CH2-CH3 and pSecVL-CH1-CH2-CH3 plasmids. Revelation was
performed as in FIG. 2. C: Immunofluorescence analysis of Cos cells
transfected with pSec-VH-CH1-CH2-CH3 and pSec-VL-CH1-CH2-CH3;
revelation with rabbit anti-VH/VL antibody plus anti-PE.
Magnification: 20.times.. D: Activity ELISA of supernatants of Cos
cell co-transfected with pSecVH-CH1-CH2-CH3 and pSecVL-CH1-CH2-CH3.
Revelation was performed as in FIG. 3.
FIG. 8: Amino acid sequence of Mono28Fc with IgG2 hinge and IgG4
CH2CH3 domains.
A: VH-CH2CH3 chain: Underlined: VH domain. Bold: linker. Double
underlining: IgG4 CH2-CH3 domains.
B: VL-CH2CH3 chain: Underlined: VL domain. Bold: linker. Double
underlining: IgG4 CH2-CH3 domains.
EXAMPLE 1: CONSTRUCTION OF THE MONOVALENT ANTIBODY MONO28FC
The CH2-CH3 domains of a human IgG1 gene (NCBI Accession BC018747)
was amplified using the following primers introducing NheI/XbaI
sites: CH2CH3-5':
TABLE-US-00001 CH2CH3-5': (SEQ ID NO: 6)
5'-ATATGCTAGCCCAGCACCTGAACTCCTG-3'; CH2CH3-3': (SEQ ID NO: 7)
5'-ATATTCTAGATTATTTACCCGGAGA-3'.
The resulting fragment was introduced into the pSC-A vector
(Stratagene, Amsterdam, The Netherlands), resulting in the
pSC-A-CH2-CH3 vector.
VH and VL domains corresponding to the CD28.3 antibody anti-human
CD28 were amplified from the previously described CNCM 1-2762 scFv
cDNA (VANHOVE et al., Blood, 102, 564-70, 2003) and NheI cloning
sites were introduced by PCR with the following primers: VH:
TABLE-US-00002 VH: Hc28.3-5': (SEQ ID NO: 8)
5'-ATATGCTAGCGGATCCGATATCGTCAAGCTGCAGCAGTCA-3'; Hc28.3-3': (SEQ ID
NO: 9) 5'-ATATGCTAGCAGATGGTGCAGCCACAGTTGAGGAGACGGTGACCA T-3'; VL:
Lc28.3-5': (SEQ ID NO: 10)
5'-ATATGCTAGCGGATCCGATATCGACATCCAGATGACCCAG-3'; Lc28.3-3': (SEQ ID
NO: 11) 5'-ATATGCTAGCAGATGGTGCAGCCACAGTCCGTTTTATTTCCAGCTTG
G-3'.
The VH and VL fragments were cloned individually 5' to the CH2-CH3
domains into the NheI site of the pSC-A-CH2-CH3 vector, resulting
in VH-pSC-A-CH2-CH3 and VL-pSC-A-CH2-CH3 plasmids. The nucleotidic
and amino acid sequences of the resulting VH-CH2CH3 and VL-CH2CH3
constructs are indicated respectively on FIGS. 1A and 1B. They are
also indicated as SEQ ID NO: 1 and 3.
Each construct was then subcloned in the EcoRV restriction site of
the pSecTag2B eukaryotic pCMV-based expression plasmid (Invitrogen,
Cergy Pontoise, France), enabling a fusion at the N-terminus with
the secretion signal from the V-J2-C region of the mouse Ig
kappa-chain provided by the pSecTag2 vector. The constructs were
proofread by sequencing. The resulting expression cassettes and the
plasmids pSec-VH-Fc(CH2-CH3) and pSec-VL-Fc(CH2-CH3) containing
these constructs are schematized respectively on FIGS. 1C and
1D.
EXAMPLE 2: EUCARYOTIC EXPRESSION OF MONO28FC
COS cells were transfected separately with pSec-VH-Fc(CH2-CH3)
(VH-Fc) or pSec-VL-Fc(CH2-CH3) (VL-Fc), or co-transfected with
pSec-VH-Fc(CH2-CH3) and pSec-VL-Fc(CH2-CH3) or, as a control,
transfected with a plasmid coding for an irrelevant green
fluorescent protein (GFP), using the Fugene lipofection kit (Roche
Diagnostics, Basel, Switzerland) according to the manufacturer's
instructions. Cultures were maintained for 3 days at 37.degree. C.,
divided one third, and put back into culture for an additional 3
days, after which time the cell supernatants were collected,
electrophoresed in 10% polyacrylamide gels and blotted onto
nitrocellulose membranes.
Blots were revealed with rabbit anti-CD28.3VH/VL (1:5000 dilution)
and an HRP-conjugated donkey antirabbit Ig antibody (Jackson
Immuno-Research Laboratories) and developed by chemiluminescence
(Amersham Pharmacia Biotech).
The results are shown on FIG. 2. Immunoreactive proteins of the
expected molecular weight (42 KDa for VL-CH2-CH3 and 44 KDa for
VH-CH2-CH3 under reducing conditions) could be observed in the cell
supernatant. A parallel analysis with non-reducing conditions
indicated an apparent molecular weight compatible with the
formation of both homodimers and heterodimers.
EXAMPLE 3: DETECTION OF MONO28FC BINDING ACTIVITY BY ELISA
Recombinant human CD28 (R&D Systems, Abingdon, United Kingdom)
was used at 1 .mu.g/mL in borate buffer (pH 9.0) to coat 96-well
microtiter plates (Immulon, Chantilly, Va.) overnight at 4.degree.
C. These immobilized CD28 target molecules will bind only
immunoreactive molecules with anti-CD28 activity.
Reactive sites were blocked with 5% skimmed milk in PBS for 2 hours
at 37.degree. C. and supernatants from control cells transfected
with the plasmid coding for GFP, from cells transfected with only
one of the plasmids pSec-VH-Fc(CH2-CH3) or pSec-VL-Fc(CH2-CH3), and
from cells co-transfected with pSec-VH-Fc(CH2-CH3) and
pSec-VL-Fc(CH2-CH3) were added at different dilutions and reacted
for 2 hours at 37.degree. C. Bound Fc fusion proteins with anti-28
activity were revealed with successive incubations (1 hour,
37.degree. C.) with rabbit anti-CD28.3VH/VL (1:2000 dilution;
custom preparation at Agrobio, Orleans, France) and horseradish
peroxidase (HRP)--conjugated donkey antirabbit Ig antibodies (1:500
dilution; Jackson ImmunoResearch Laboratories, Bar Harbor, Me.).
Bound antibody was revealed by colorimetry using the TMB substrate
(Sigma, L'Isle d'Abeau Chesnes, France) read at 450 nm.
The results are shown on FIG. 3 A.
Supernatants from control cells (transfected with the plasmid
coding for GFP) or from cells transfected with only one of the
plasmids pSec-VH-Fc(CH2-CH3) or pSec-VL-Fc(CH2-CH3) did not contain
any detectable level of immunoreactive molecule. This indicated
that VH-Fc or VL-Fc homodimers cannot bind CD28. In contrast,
supernatants from cells co-transfected with pSec-VH-Fc(CH2-CH3) and
pSec-VL-Fc(CH2-CH3) contained dilution-dependant levels of
immunoreactive molecules.
Mono28Fc was purified from culture supernatants of COS cells
co-transfected with pSec-VH-Fc(CH2-CH3) and pSec-VL-Fc(CH2-CH3) and
maintained for 3 days at 37.degree. C.
Supernatants were passed through G-Protein Sepharose columns
(Amersham) at a rate of 1 ml/min. The columns were rinsed with PBS
and proteins were eluted with glycine buffer (pH 2.8), concentred
by osmotic water retrieval using polyethylene glycol (Fluka,
Riedel-de Haen, Germany) and dialysed extensively against PBS at
4.degree. C.
After purification, the Mono28Fc molecules were tested by ELISA as
described above. The results are shown on FIG. 3B.
These results show that 50% of the binding activity to CD28 could
be reached at a concentration of 100 ng/ml, which represents 1.16
nM.
EXAMPLE 4: DETECTION OF MONO28FC BINDING ACTIVITY BY FLOW
CYTOMETRY
The binding of Mono28Fc was confirmed by flow cytometry using CD28+
Jurkat human T cells, which express CD28, or on Raji cells, a human
B cell line that does not express CD28.
Jurkat T cells or Raji cells were incubated for 1 hour at 4.degree.
C. with purified Mono28Fc proteins or with Fab fragments of CD28.3
(VANHOVE et al., Blood, 102, 564-70, 2003), at 10 .mu.g/ml for 30
min. As a control, cells were incubated with rabbit anti-VII/VH
antibodies plus FITC-labeled goat anti-rabbit immunoglobulins only.
Bound Fc fusion monomers were detected with a rabbit
anti-CD28.3VH/VL and a fluorescein isothiocyanate
(FITC)--conjugated donkey anti-rabbit Ig antibody (dilution 1:200;
Jackson ImmunoResearch Laboratories) for 30 minutes at 4.degree. C.
Cells were then analyzed by fluorescence-activated cell sorting
(FACS).
The results are shown on FIG. 4. Both mono28Fc and the Fab fragment
of CD28.3 bind Jurkat T cells. In contrast, no binding of the
mono28Fc protein could be observed on Raji cells, a human B cell
line that does not express CD28. These data demonstrate mono28Fc
that binds specifically to CD28.sup.+ cells.
EXAMPLE 5: MONO28FC HAS NO AGONIST ACTIVITY ON HUMAN T CELLS
To verify that mono28Fc binds to CD28 and does not induce
activation of the target T cell, we compared the biological effect
of Mono28Fc with those of the superagonistic antibody ANC28.1
(WAIBLER et al., PLoS ONE, 3, e1708, 2008), or of sc28AT, a
monovalent anti-CD28 ligand without Fc domain (VANHOVE et al.,
Blood, 102, 564-70, 2003).
Human PBMC (10.sup.5/well) were cultivated in culture medium
without additive (control), or in culture medium with 10 .mu.g/ml
of mono28Fc, of sc28AT, or of ANC28.1 for 3 days. 0.5 .mu.Ci
.sup.3H-tymidine was added for the last 16 h of the culture.
Incorporated radioactivity was evaluated on a scintillation counter
after transfer on nitrocellulose membranes. The results are shown
on FIG. 5.
As expected, ANC28.1 induced a robust proliferation of the target
cells. In contrast, Mono28Fc, as well as sc28AT did not induce any
response in this assay.
EXAMPLE 6: PHARMACOKINETICS OF MONO28FC IN MICE
Recombinant proteins fused with an Fc fragment and immunoglobulins
usually present an extended half-life in vivo because they are
recognised by the FcRn receptor presented on endothelial and
epithelial cells allowing the recycling of that molecules back in
the circulation. To determine if our Mono28FC molecule also
presents an extended half-live, we followed the distribution in
mice of Mono28Fc in comparison with monovalent Fab 28.3 antibody
fragments and native IgG CD28.3 antibodies.
Each protein tested (288 .mu.g per injection) was injected into the
tail vein of male Swiss mice. Blood samples (2 .mu.L) were
collected at different times from the tail vein. The proteins were
quantified by measuring the CD28 binding activity in blood samples
by ELISA. The data were analyzed by Siphar software (Simed,
Utrecht, The Netherlands) with the use of a 2-compartment model.
Significance was evaluated with an non-parametric ANOVA test
followed by a Bonferroni's Multiple Comparison Test.
The results are shown on FIG. 6.
The distribution half-live (T.sub.1/2.alpha.) was of 2.5.+-.1.1;
5.1.+-.0.3 and 5.4.+-.1.2 hours for IgG, Fab and Mono28Fc,
respectively. The elimination half-live (T.sub.1/2.beta.) was of
119.+-.19; 39.+-.6 and 83.+-.26 hours for IgG, Fab and Mono28Fc,
respectively (FIG. 6). The data reveal a significant increase of
the elimination half-live of Mono28Fc, as compared with Fab
fragments, whereas no statistical difference is pointed out when
Mono28Fc is compared with a divalent IgG.
EXAMPLE 7: COMPARISON OF MONO28FC WITH A CONSTRUCTION COMPRISING
THE CH1-CH2-CH3 IG HEAVY CHAIN DOMAINS
The human IgG1 CH1-CH2-CH3 cDNA was given by Dr. S. Birkle (Univ.
Nantes, France). It was inserted into the pcDNA3.1 into the
HindIII/BamHI restriction sites, resulting in the
pcDNA3.1-CH1-CH2-CH3 plasmid. VH and VL domains corresponding to
the CD28.3 antibody anti-human CD28 (NUNES et al., Int Immunol, 5,
311-5, 1993) were amplified as described in Example 1 above,
digested with the NheI enzyme and inserted separately into the NheI
site of the pcDNA3.1-CH1-CH2-CH3 plasmid. The VH-CH1-CH2-CH3 and
VL-CH1-CH2-CH3 cassettes were then excised by EcoRV/XbaI digestion
and inserted into the EcoR V digested pSecTag2B vector
(Invitrogen), as disclosed in Example 1.
After transfection in Cos cells, messenger RNA molecules
corresponding to the two chains were equally synthesised (FIG. 7A).
The analysis of proteins by western blotting revealed the synthesis
of some corresponding molecules, although clearly more abundant
within the cell (FIG. 7B, left panel) than in the supernatant (FIG.
7B, right panel) for the light chain (VL-CH1-CH2-CH3). By
immunohistology, the synthesis of both heavy and light chains by
transfected Cos cells could be confirmed (FIG. 7C). By ELISA, no
CD28 binding activity could be detected in the supernatant (data
not shown) nor in transfected cell lysates (FIG. 7D).
SEQUENCE LISTINGS
1
1411041DNAArtificial SequenceChimeric constructCDS(1)..(1041) 1gtc
aag ctg cag cag tca gga gct gag ctg gtg aaa ccc ggg gcg tcg 48Val
Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser1 5 10
15gtg agg ctg tcc tgc aag gcg tct ggt tac acc ttc act gaa tat att
96Val Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Ile
20 25 30ata cac tgg ata aag ctg agg tct gga cag ggt ctt gag tgg att
ggg 144Ile His Trp Ile Lys Leu Arg Ser Gly Gln Gly Leu Glu Trp Ile
Gly 35 40 45tgg ttt tac cct gga agt aat gat ata cag tac aat gcg aaa
ttc aag 192Trp Phe Tyr Pro Gly Ser Asn Asp Ile Gln Tyr Asn Ala Lys
Phe Lys 50 55 60ggc aag gcc aca ttg act gcg gac aaa tcc tcc agc acc
gtc tat atg 240Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr
Val Tyr Met65 70 75 80gaa ctt act gga ttg aca tct gag gac tct gcg
gtc tat ttc tgt gca 288Glu Leu Thr Gly Leu Thr Ser Glu Asp Ser Ala
Val Tyr Phe Cys Ala 85 90 95aga cgc gac gat ttc tct ggt tac gac gcc
ctt cct tac tgg ggc caa 336Arg Arg Asp Asp Phe Ser Gly Tyr Asp Ala
Leu Pro Tyr Trp Gly Gln 100 105 110ggg acc atg gtc acc gtc tcc tca
act gtg gct gca cca tct gct agc 384Gly Thr Met Val Thr Val Ser Ser
Thr Val Ala Ala Pro Ser Ala Ser 115 120 125cca gca cct gaa ctc ctg
ggg gga ccg tca gtc ttc ctc ttc ccc cca 432Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 130 135 140aaa ccc aag gac
acc ctc atg atc tcc cgg acc cct gag gtc aca tgc 480Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys145 150 155 160gtg
gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg 528Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 165 170
175tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag
576Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
180 185 190gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc
gtc ctg 624Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 195 200 205cac cag gac tgg ctg aat ggc aag gag tac aag tgc
aag gtc tcc aac 672His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 210 215 220aaa gcc ctc cca gcc ccc atc gag aaa acc
atc tcc aaa gcc aaa ggg 720Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly225 230 235 240cag ccc cga gaa cca cag gtg
tac acc ctg ccc cca tcc cgg gag gag 768Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu 245 250 255atg acc aag aac cag
gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat 816Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 260 265 270ccc agc gac
atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac 864Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 275 280 285aac
tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc 912Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 290 295
300ctc tat agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac
960Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn305 310 315 320gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac
aac cac tac acg 1008Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 325 330 335cag aag agc ctc tcc ctg tct ccg ggt aaa
taa 1041Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340
3452346PRTArtificial SequenceSynthetic Construct 2Val Lys Leu Gln
Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser1 5 10 15Val Arg Leu
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Ile 20 25 30Ile His
Trp Ile Lys Leu Arg Ser Gly Gln Gly Leu Glu Trp Ile Gly 35 40 45Trp
Phe Tyr Pro Gly Ser Asn Asp Ile Gln Tyr Asn Ala Lys Phe Lys 50 55
60Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr Met65
70 75 80Glu Leu Thr Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
Ala 85 90 95Arg Arg Asp Asp Phe Ser Gly Tyr Asp Ala Leu Pro Tyr Trp
Gly Gln 100 105 110Gly Thr Met Val Thr Val Ser Ser Thr Val Ala Ala
Pro Ser Ala Ser 115 120 125Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 130 135 140Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys145 150 155 160Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 165 170 175Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 180 185 190Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 195 200
205His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
210 215 220Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly225 230 235 240Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu 245 250 255Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 260 265 270Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 275 280 285Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 290 295 300Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn305 310 315
320Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
325 330 335Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340
34531005DNAArtificial SequenceChimeric constructCDS(1)..(1005) 3gac
atc cag atg acc cag tct cca gcc tcc cta tct gtt tct gtg gga 48Asp
Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly1 5 10
15gaa act gtc acc atc acg tgt cga aca aat gaa aat att tac agt aat
96Glu Thr Val Thr Ile Thr Cys Arg Thr Asn Glu Asn Ile Tyr Ser Asn
20 25 30tta gca tgg tat cag cag aaa cag gga aaa tct cct cag ctc ctg
atc 144Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu
Ile 35 40 45tat gct gca aca cac tta gta gag ggt gtg cca tca agg ttc
agt ggc 192Tyr Ala Ala Thr His Leu Val Glu Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60agt gga tca ggc aca cag tat tcc ctc aag atc acc agc
ctg cag tct 240Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser
Leu Gln Ser65 70 75 80gaa gat ttt ggg aat tat tac tgt caa cac ttt
tgg ggt act ccg tgc 288Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His Phe
Trp Gly Thr Pro Cys 85 90 95acg ttc gga ggg ggg acc aag ctg gaa ata
aaa cgg act gtg gct gca 336Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110cca tct gct agc cca gca cct gaa
ctc ctg ggg gga ccg tca gtc ttc 384Pro Ser Ala Ser Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe 115 120 125ctc ttc ccc cca aaa ccc
aag gac acc ctc atg atc tcc cgg acc cct 432Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 130 135 140gag gtc aca tgc
gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 480Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val145 150 155 160aag
ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca 528Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 165 170
175aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc
576Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
180 185 190ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac
aag tgc 624Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys 195 200 205aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag
aaa acc atc tcc 672Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser 210 215 220aaa gcc aaa ggg cag ccc cga gaa cca cag
gtg tac acc ctg ccc cca 720Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro225 230 235 240tcc cgg gag gag atg acc aag
aac cag gtc agc ctg acc tgc ctg gtc 768Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val 245 250 255aaa ggc ttc tat ccc
agc gac atc gcc gtg gag tgg gag agc aat ggg 816Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 260 265 270cag ccg gag
aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac 864Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 275 280 285ggc
tcc ttc ttc ctc tat agc aag ctc acc gtg gac aag agc agg tgg 912Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 290 295
300cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac
960Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His305 310 315 320aac cac tac acg cag aag agc ctc tcc ctg tct ccg
ggt aaa taa 1005Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 3304334PRTArtificial SequenceSynthetic Construct 4Asp Ile
Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly1 5 10 15Glu
Thr Val Thr Ile Thr Cys Arg Thr Asn Glu Asn Ile Tyr Ser Asn 20 25
30Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45Tyr Ala Ala Thr His Leu Val Glu Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Leu
Gln Ser65 70 75 80Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His Phe Trp
Gly Thr Pro Cys 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110Pro Ser Ala Ser Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe 115 120 125Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro 130 135 140Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val145 150 155 160Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 165 170
175Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
180 185 190Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys 195 200 205Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser 210 215 220Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro225 230 235 240Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val 245 250 255Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 260 265 270Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 275 280 285Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 290 295
300Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His305 310 315 320Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 325 33056PRTArtificial SequencePeptide linker 5Thr Val Ala
Ala Pro Ser1 5628DNAArtificial SequencePCR primer 6atatgctagc
ccagcacctg aactcctg 28725DNAArtificial SequencePCR primer
7atattctaga ttatttaccc ggaga 25840DNAArtificial SequencePCR primer
8atatgctagc ggatccgata tcgtcaagct gcagcagtca 40946DNAArtificial
SequencePCR primer 9atatgctagc agatggtgca gccacagttg aggagacggt
gaccat 461040DNAArtificial SequencePCR primer 10atatgctagc
ggatccgata tcgacatcca gatgacccag 401148DNAArtificial SequencePCR
primer 11atatgctagc agatggtgca gccacagtcc gttttatttc cagcttgg
481212PRTHomo sapiens 12Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys
Pro1 5 1013349PRTArtificial SequenceChimeric protein 13Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser1 5 10 15Val Arg
Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Ile 20 25 30Ile
His Trp Ile Lys Leu Arg Ser Gly Gln Gly Leu Glu Trp Ile Gly 35 40
45Trp Phe Tyr Pro Gly Ser Asn Asp Ile Gln Tyr Asn Ala Lys Phe Lys
50 55 60Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr
Met65 70 75 80Glu Leu Thr Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr
Phe Cys Ala 85 90 95Arg Arg Asp Asp Phe Ser Gly Tyr Asp Ala Leu Pro
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala Glu Arg
Lys Cys Cys Val Glu Cys 115 120 125Pro Pro Cys Pro Ala Pro Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu 130 135 140Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu145 150 155 160Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 165 170 175Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 180 185
190Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu
195 200 205Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys 210 215 220Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
Thr Ile Ser Lys225 230 235 240Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser 245 250 255Gln Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys 260 265 270Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 275 280 285Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 290 295 300Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln305 310
315 320Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn 325 330 335His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
340 34514337PRTArtificial SequenceChimeric protein 14Asp Ile Gln
Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly1 5 10 15Glu Thr
Val Thr Ile Thr Cys Arg Thr Asn Glu Asn Ile Tyr Ser Asn 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile 35 40
45Tyr Ala Ala Thr His Leu Val Glu Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Leu Gln
Ser65 70 75 80Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His Phe Trp Gly
Thr Pro Cys 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
Glu Arg Lys Cys 100 105 110Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro 115 120 125Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser 130 135 140Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser Gln Glu Asp145 150 155 160Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 165 170 175Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 180
185
190Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
195 200 205Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys 210 215 220Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr225 230 235 240Leu Pro Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr 245 250 255Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 260 265 270Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 275 280 285Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 290 295 300Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu305 310
315 320Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
Gly 325 330 335Lys
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